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PubMed1984

Tissue levels and purification by affinity chromatography of the calmodulin-stimulated Ca2+ -transport ATPase in pig antrum smooth muscle.

The Ca2+ -transport ATPase [Ca2+ + Mg2+)-ATPase) in a plasma membrane-rich fraction of porcine antrum (stomach) smooth muscle, is stimulated 2.9-times by calmodulin in the presence of 0.2 mg/ml saponin and reaches a value of 12.0 +/- 2.0 (4) mumol/100 mg protein (equivalent to 110 g wet tissue) per min at 37 degrees C and 10(-5) M [Ca2+]. Saponin was found to specifically potentiate the calmodulin-(Ca2+ + Mg2+)-ATPase interaction, even in the Triton X-100 solubilized enzyme. The conditions for purification of the (Ca2+ + Mg2+)-ATPase by affinity chromatography on a calmodulin-Sepharose 4B gel were optimized. The purified enzyme has a specific activity of 11.9 mumol/mg protein per min at 37 degrees C, 10(-5) M [Ca2+], 0.6 microM calmodulin, and shows a double polypeptide band at 140 and 150 kDa. The (Ca2+ + Mg2+)-ATPase can be incorporated in artificial liposomes that thereupon show an ATP-dependent Ca2+ uptake (Ca:ATP = 1.0). The magnitude of the calmodulin stimulation of the isolated enzyme depends on its phospholipid environment. When isolated in the presence of phosphatidylserine no calmodulin stimulation is observed. After reconstitution in phosphatidylcholine the calmodulin stimulation amounts to 4.05 +/- 0.63 (n = 12) times.

Animals↗

PubMed1983

Trifluoperazine, a calmodulin antagonist, inhibits muscle cell fusion.

We investigated the effect of trifluoperazine (TFP), a calmodulin antagonist, on the fusion of chick skeletal myoblasts in culture. TFP was found to inhibit myoblast fusion. This effect occurs at concentrations that have been reported to inhibit Ca2+-calmodulin in vitro, and is reversed upon removal of TFP. In addition, other calmodulin antagonists, including chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W7), and N-(6-aminohexyl)-1-naphthalene-sulfonamide (W5), inhibit fusion at doses that correspond closely to the antagonistic effects of these drugs on calmodulin. The expression of surface acetylcholine receptor, a characteristic aspect of muscle differentiation, is not impaired in TFP-arrested myoblasts. Myoblasts inhibited from fusion by 10 microM TFP display impaired alignment. In the presence of the Ca2+ ionophore A23187, the fusion block by 10 microM TFP is partially reversed and myoblast alignment is restored. The presence and distribution of calmodulin in both prefusional myoblasts and fused muscle cells was established by immunofluorescence. We observed an apparent redistribution of calmodulin staining that is temporally correlated with the onset of myoblast fusion. Our findings suggest a possible role for calmodulin in the regulation of myoblast fusion.

Animals↗

PubMed1993

Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings).

The effects of calmodulin and of controlled trypsin treatments on the activity of the Ca2+ pump were investigated in plasma membrane purified from radish (Raphanus sativus L.) seedlings. Treatment of the plasma membrane with ethylenediaminetetra-acetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Ca2+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Ca2+ pump activity was maximal at low free Ca2+ (2-5 [mu]M) and decreased with the increase of free Ca2+ concentration. The Ca2+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the Ca2+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free Ca2+ concentration in the assay medium. Moreover, the Ca2+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.

Journal Article↗

PubMed1987

A calcium-dependent but calmodulin-independent protein kinase from soybean.

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar ( approximately 2 micromolar). The protein kinase activity was stimulated 100-fold by >/=10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (</=2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound (45)Ca(2+) in the presence of KCl and MgCl(2), which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.

Journal Article↗

PubMed1987

Calmodulin mRNA in Barley (Hordeum vulgare L.) : Apparent Regulation by Cell Proliferation and Light.

Calmodulin is encoded by a 650-nucleotide mRNA in higher plants. This messenger was identified in barley and pea by a combination of in vitro translation and blot hybridization experiments using anti-sense RNA produced from an eel calmodulin cDNA probe. In all plant tissues tested, calmodulin mRNA represents between 0.01 and 0.1% of the total translatable mRNA population. Calmodulin mRNA levels are three- to fourfold higher in the meristematic zone of the first leaf of barley. At all other stages of leaf cell differentiation, calmodulin mRNA levels are nearly identical. During light-induced development in barley leaves, the relative proportion of translatable calmodulin mRNA declines about twofold. Cytoplasmic mRNAs that may encode calmodulin-like proteins were also detected. The levels of several of these putative Ca(2+)-binding protein mRNAs are modulated during the course of light-induced barley leaf cell development.

Journal Article↗

PubMed1987

Cardiac calmodulin-stimulated protein phosphatase: purification and identification of specific sarcolemmal substrates.

A calmodulin-stimulated protein phosphatase has been purified from bovine myocardium. The purification procedure involves sequential DEAE-Sephacel ion exchange chromatography, calmodulin-Sepharose affinity chromatography, and high performance liquid chromatography using a Spherogel TSK DEAE 5PW column. By SDS polyacrylamide gel electrophoresis, the purified cardiac phosphatase consists of two subunits of Mr 61,000 and 19,000, similar to the brain enzyme, calcineurin. Protein phosphatase activity of the cardiac enzyme is stimulated by Ca2+-calmodulin and inhibited by the calmodulin antagonist drug, calmidazolium. Effects of a series of divalent cations on catalytic activity of the cardiac calmodulin-stimulated protein phosphatase are similar to those observed with calcineurin, when the two enzymes are assayed under identical conditions. Highly enriched preparations of bovine cardiac sarcolemma contain substrates of cAMP-dependent protein kinase of Mr 166 K, 133 K, 108 K, 79 K, 39 K, and 14 K, which are specifically dephosphorylated by the calmodulin-stimulated phosphatase with pseudofirst-order rate constants of 0.23, 0.46, 0.69, 0.35, 0.69, and 0.115 min-1, respectively. These substrates are not present in purified preparations of cardiac sarcoplasmic reticulum. These results support a role of the calmodulin-stimulated phosphatase in the Ca2+-regulation of specific sarcolemmal processes by protein dephosphorylation.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1996

Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement.

Calmodulin is a calcium-binding protein that regulates a wide range of enzymes. It is also one of the few examples of a small protein capable of binding to peptides with very high affinity, and is therefore an interesting candidate for biotechnological applications and a good model system for studying how proteins associate. We have synthesized a complete series of peptides derived from the recognition sequence of skeletal muscle myosin light-chain kinase, corresponding to single-point amino acid mutations to alanine. These peptides bind to calmodulin with a biphasic kinetic: a fast association step followed by a slow intramolecular isomerisation. We have measured the isomerisation rate (k(isom)) of these peptides for calmodulin by stopped-flow analysis, and their association and dissociation kinetic constants (k(on) and k(off)) by real-time interaction analysis using surface plasmon resonance detection. In addition, k(off) constants were measured by competition experiments using a high-sensitivity luminescence analyser and native polyacrylamide gels. We have observed that all the alanine-scanning peptides bound to calmodulin with better affinity than the wild-type. In one case, a Asn-->Ala substitution resulted in a 1000-fold improvement in affinity, owing to a slower off-rate. Our results indicate that naturally occurring calmodulin binders may have evolved to have high affinities, but far from the maximum. Our affinity data are in contrast with recently published predictions of interactions responsible for high-affinity calmodulin binding based on modelling and energy calculations.

Alanine↗

PubMed1983

Calmodulin-dependent glycogen synthase kinase: identification in liver of normal and phosphorylase kinase-deficient rats.

We have purified a calmodulin-dependent glycogen synthase kinase from livers of normal and phosphorylase kinase-deficient (gsd/gsd) rats. No differences between normal and gsd/gsd rats were apparent in either (a) the ability of liver extracts to phosphorylate exogenous glycogen synthase in a Ca2+- and calmodulin-dependent manner or (b) the purification of the calmodulin-dependent synthase kinase. Although extracts from rat liver, when compared to rabbit liver extracts, had a significantly reduced ability to phosphorylate exogenous synthase, the calmodulin-dependent synthase kinase could be purified from rat liver using a protocol identical to that described for rabbit liver. Moreover, the synthase kinase purified from rat liver had properties very similar to those of the rabbit liver enzyme. The enzyme was completely dependent on calmodulin for activity against glycogen synthase, was unable to phosphorylate phosphorylase b, catalyzed the rapid incorporation of 0.4 mol phosphate/mol of glycogen synthase subunit, selectively phosphorylated sites 1b and 2 in the glycogen synthase molecule, had a Stokes' radius of about 70 A, and appeared to be composed of subunits of Mr 56,000 and 57,000. These observations led us to conclude that (1) calmodulin-dependent glycogen synthase kinase is distinct from other kinases previously described and (2) the rat liver kinase and the rabbit liver kinase are very similar enzymes.

Animals↗

PubMed1985

A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries.

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1983

The role of calmodulin in cell transformation.

Two cell lines transformed with temperature sensitive retroviruses were examined for: their ability to grow in low Ca2+ medium, their calmodulin levels and changes in calmodulin acceptor proteins. Both cell lines grow in low Ca2+ medium at the permissive temperature 34 degrees C while both lines did not replicate at the non-permissive temperature 39 degrees C. The NRKLA23 cells have nearly twice as much calmodulin at the permissive temperature than they do at the non-permissive temperature while the 6M2 cells have an equal amount of calmodulin at both temperatures. Both cell lines exhibit changes in the calmodulin acceptor proteins going from the permissive to the non-permissive temperature. We suspect that the changes in the calmodulin acceptor proteins may be involved in the altered Ca2+-sensitivity of growth in the cells going from the permissive to non-permissive temperature.

Animals↗

PubMed1992

Calmodulin is involved in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary chromaffin cells.

The role of calmodulin in exocytotic secretion was studied using digitonin-permeabilized bovine adrenal medullary chromaffin cells. Addition of calmodulin to the permeabilized cells increased Ca(2+)-dependent norepinephrine release in a dose-dependent manner. Unlike calmodulin, addition of caldesmon, actin or bovine serum albumin did not increase the release. Calmodulin increased the release at Ca2+ concentrations of more than 10(-6) M and its effect increased with increase in Mg2+ concentration. Th release of norepinephrine enhanced by calmodulin was inhibited by tetanus toxin, which specifically inhibits exocytotic secretion. These results indicate directly that calmodulin plays an important role in exocytotic secretion from chromaffin cells.

Actins↗

PubMed1992

1,25-Dihydroxyvitamin D effects in the kidney: induction of calmodulin binding proteins.

A marked induction of 125I-calmodulin binding proteins (185kDa and 115kDa) occurred in the rat kidney in response to treatment with 1,25-dihydroxyvitamin D3 (100 ng/day s.c. x 7d). These 125I-calmodulin binding proteins, measured by the gel overlay procedure, exhibited calcium dependence and were abolished in the presence of excess unlabelled calmodulin. The response was tissue specific: there was no change in 125I-calmodulin binding in rat testis, heart, and brain and only a modest elevation of binding to one calmodulin binding protein in the intestinal mucosa. These results are particularly important in suggesting that the calmodulin signal transduction mechanism may, via changes in its acceptor proteins, participate in mediating some biological effects of 1,25-dihydroxyvitamin D3.

Animals↗

PubMed1988

An altered calmodulin binding protein in cystic fibrosis--a clue to the biochemical defect.

Cytosolic extracts prepared from submandibular tissues of CF patients showed a greater ability to activate calmodulin-deficient cyclic AMP phosphodiesterase than did control extracts. Thus, apparent calmodulin levels measured by cyclic AMP phosphodiesterase activation were significantly greater (p less than 0.001) in CF submandibular extracts than control; whereas calmodulin levels measured by radioimmunoassay were not different. In addition a calmodulin-binding protein of molecular weight 61,000 which showed a specific Ca2+-dependent interaction with calmodulin, was shown to be markedly altered in heat-treated extracts from CF submandibular glands. The results indicate that a specific protein which modulates selective biological action(s) of calmodulin is altered in CF. This would provide a biochemical link between disturbances in autonomic function and Ca2+ homeostasis seen in this disease and might therefore be closely related to the genetic defect.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1996

A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase.

HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide synthase.

Acetylcholine↗

PubMed1990

Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin.

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.

Amino Acid Oxidoreductases↗

PubMed1994

Dual role of calmodulin in autophosphorylation of multifunctional CaM kinase may underlie decoding of calcium signals.

Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase makes it Ca2+ independent by trapping bound calmodulin and by enabling the kinase to remain partially active even after calmodulin dissociates. We show that autophosphorylation is an intersubunit reaction between neighbors in the multimeric kinase which requires two molecules of calmodulin. Ca2+/calmodulin acts not only to activate the "kinase" subunit but also to present effectively the "substrate" subunit for autophosphorylation. Conversion of the kinase to the potentiated or trapped state is a cooperative process that is inefficient at low occupancy of calmodulin. Simulations show that repetitive Ca2+ pulses at limiting calmodulin lead to the recruitment of calmodulin to the holoenzyme, which further stimulates autophosphorylation and trapping. This cooperative, positive feedback loop will potentiate the response of the kinase to sequential Ca2+ transients and establish a threshold frequency at which the enzyme becomes highly active.

Amino Acid Sequence↗

PubMed1998

Lack of calmodulin antagonism of melatonin in T-lymphocyte activation.

Despite various reported effects of the pineal hormone melatonin on the immune system, its mechanism of action on immune cells is still unknown. Since melatonin has been suggested as a physiological antagonist to calmodulin in certain cell types, we investigated effects of melatonin on calmodulin-dependent IL-2 production and proliferation of activated T-lymphocytes. It was found, however, that, in contrast to the calmodulin antagonists trifluoperazine and W7, melatonin neither inhibited the IL-2 production of activated lymphoblastoid Jurkat T-cells nor decreased the mitogen response of peripheral blood mononuclear leukocytes. Preincubation of Jurkat cells with melatonin did not influence trifluoperazine effects on IL-2 production indicating that melatonin does not bind to the same sites of calmodulin as trifluoperazine, as has been postulated. In conclusion, these results did not give any evidence for a calmodulin antagonism of melatonin in T-lymphocyte activation. Thus, melatonin as a calmodulin antagonist appears not to be a universal phenomenon.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1996

Calmodulin is the divalent cation receptor for rapid endocytosis, but not exocytosis, in adrenal chromaffin cells.

Exocytosis and the ensuing rapid endocytosis in adrenal chromaffin cells are both Ca(2+)-dependent phenomena but differ in their divalent cation specificity, implying distinct Ca2+ receptors for the two processes. To ascertain whether calmodulin is the Ca2+ receptor for either process, we blocked its function by introducing calmodulin-binding peptides or anti-calmodulin antibodies into these cells. Exo/endocytosis was followed by measurement of cell membrane capacitance. Rapid endocytosis, but not exocytosis, was abolished by these treatments, indicating that calmodulin is the Ca2+ receptor for rapid endocytosis but is not involved in exocytosis. The principal calmodulin target is not protein phosphatase-2B, as antagonism of this enzyme did not inhibit but accelerated rapid endocytosis. Calmodulin may thus regulate both the rate and extent of rapid endocytosis by distinct pathways.

Adrenal Medulla↗